Giardia duodenalis-induced G0/G1 intestinal epithelial cell cycle arrest and apoptosis involve activation of endoplasmic reticulum stress in vitro

Abstract

Giardia duodenalis is a zoonotic intestinal protozoan parasite that may cause host diarrhea and chronic gastroenteritis, resulting in great economic losses annually and representing a significant public health burden across the world. However, thus far, our knowledge on the pathogenesis of Giardia and the related host cell responses is still extensively limited. The aim of this study is to assess the role of endoplasmic reticulum (ER) stress in regulating G0/G1 cell cycle arrest and apoptosis during in vitro infection of intestinal epithelial cells (IECs) with Giardia. The results showed that the mRNA levels of ER chaperone proteins and ER-associated degradation genes were increased and the expression levels of the main unfolded protein response (UPR)-related proteins (GRP78, p-PERK, ATF4, CHOP, p-IRE1, XBP1s and ATF6) were increased upon Giardia exposure. In addition, cell cycle arrest was determined to be induced by UPR signaling pathways (IRE1, PERK and ATF6) through upregulation of p21 and p27 levels and promotion of E2F1-RB complex formation. Upregulation of p21 and p27 expression was shown to be related to Ufd1-Skp2 signaling. Therefore, the cell cycle arrest was induced by ER stress when infected with Giardia. Furthermore, the apoptosis of the host cell was also assessed after exposure to Giardia. The results indicated that apoptosis would be promoted by UPR signaling (PERK and ATF6), but would be suppressed by the hyperphosphorylation of AKT and hypophosphorylation of JNK that were modulated by IRE1 pathway. Taken together, both of the cell cycle arrest and apoptosis of IECs induced by Giardia exposure involved the activation of the UPR signaling. The findings of this study will deepen our understanding of the pathogenesis of Giardia and the associated regulatory network.

Keywords: ER stress; Giardia duodenalis; IECs; UPR; apoptosis; cell cycle arrest.

Figure 1

Giardia reduced host cell viability and induced G0/G1 cell cycle arrest and apoptosis. (A) Inhibition of cell viability by Giardia exposure in Caco-2 and HT-29 cells. Cell viability was detected via CCK-8 assay (n = 5 wells/group). Cells to Giardia ratio was 1:3, 1:5 or 1:10. (B) PI staining followed by flow cytometry was used to detect cell cycle distribution after the exposure of Giardia with 0, 3, 6, 12 h in Caco-2 and HT-29 cells. G0/G1, S and G2 indicate the different phases of the cell cycle. (C) The qPCR analysis (n = 3) was used to measure the mRNA levels of cyclins and CDKs with Giardia exposure in Caco-2 and HT-29 cells. The relative amounts of mRNA were normalized against GAPDH mRNA and expressed relative to the mRNA abundance in control. (D) Western blot analysis was used to measure the protein levels of cyclins and CDKs in Caco-2 and HT-29 cells treated with Giardia. (E) Western blotting was used to measure the expression levels of cleaved CASP3 and cleaved PARP in Caco-2 and HT-29 cells treated by Giardia. (F) Apoptotic effects of Giardia exposure on Caco-2 and HT-29 cells as assessed by AO/EB staining (n = 3 wells/group, scale bar = 1000 μm). The fluorescence intensity was quantified using Image J. All experiments were repeated at least three times. Data were presented as mean ± SD. *p < 0.05, **p < 0.01 versus relative control group. (D, E) Signal intensity of protein band was analyzed by using Image J software. The results of western blot analyses (n = 3) were normalized against the level of β-actin. (B, D–F) Representative pictures are shown.

Figure 2

The expressions of p21 and p27 were increased and E2F1-RB complex formation was promoted by Giardia exposure. (A) The mRNA levels of E2F1-E2F8 with Giardia exposure in Caco-2 and HT-29 cells were measured by qPCR analysis (n = 3). (B) The protein levels of E2F1-E2F4 in Caco-2 and HT-29 cells treated with Giardia were analyzed by western blot analysis. (C) The E2F1-RB complex was detected by co-IP. Both IP and Input samples were analyzed by SDS-PAGE and immunoblotting. (D) The qPCR analysis (n = 3) was used to measure the mRNA levels of p21, p27 and p53 with Giardia exposure in Caco-2 and HT-29 cells. (E) Western blot analysis was used to measure the protein levels of p21, p27 and p53 with Giardia exposure in Caco-2 and HT-29 cells. All experiments were repeated at least three times. Data were presented as mean ± SD. *p < 0.05, **p < 0.01 versus relative control group. (A, D) The relative amounts of mRNA were normalized against GAPDH mRNA and expressed relative to the mRNA abundance in control. (B, C, E) Representative images are presented. The results of western blot analyses (n = 3) were normalized against the level of β-actin.

Figure 3

The UPR and ERAD pathways were activated by Giardia exposure. (A) The mRNA levels of ER stress-related genes and ERAD-related genes with Giardia exposure in Caco-2 and HT-29 cells were analyzed by qPCR analysis (n = 3). The relative amounts of mRNA were normalized against GAPDH mRNA and expressed relative to the mRNA abundance in control. (B) Western blot analysis (n = 3) was used to measure the expression levels of ER stress-related proteins with Giardia exposure in Caco-2 and HT-29 cells. Signal intensity of protein band was analyzed by using Image J software. The results of western blot analyses were normalized against the level of β-actin. All experiments were repeated at least three times. Data were presented as mean ± SD. *p < 0.05, **p < 0.01 versus relative control group.

Figure 4

Giardia arrested cell cycle by activating three UPR signaling pathways. (A) PI staining followed by flow cytometry was used to detect cell cycle distribution in Caco-2 cells treated with or without inhibitors accompanied by Giardia exposure for 6 h; IRE1 inhibitor (MKC3946), PERK inhibitor (GSK2606414) and ATF6 inhibitor (AEBSF). G0/G1, S, and G2 indicate the different phases of the cell cycle. Under treatment of the combination of Giardia with (B) inhibitor MKC3946, (C) GSK2606414 or (D) AEBSF in Caco-2 cells, the expressions of cyclins, CDKs and E2Fs were detected by western blotting. (E) Co-IP experiments were performed to detect E2F1-RB complex formation in Caco-2 cells treated with or without inhibitors accompanied by Giardia exposure for 6 h; IRE1 inhibitor (MKC3946), PERK inhibitor (GSK2606414) and ATF6 inhibitor (AEBSF). Gl, Giardia. All experiments were repeated at least three times. Data were presented as mean ± SD. *p < 0.05, **p < 0.01 versus relative control group. (B–E) Signal intensity of protein band was analyzed by using Image J software. The results of western blot analyses (n = 3) were normalized against the level of β-actin. (A–E) Representative images are presented.

Figure 5

The expressions of p21 and p27 were enhanced by Giardia exposure via activating UPR pathways. (A) Western blot analysis (n = 3) was used to measure the protein levels of p21 and p27 in Caco-2 cells treated with or without inhibitors accompanied by Giardia exposure for 6 h; IRE1 inhibitor (MKC3946), PERK inhibitor (GSK2606414) and ATF6 inhibitor (AEBSF). The results of western blot analyses were normalized against the level of β-actin. Gl, Giardia. (B) The qPCR analysis was used to detect the mRNA levels of Ufd1 and Skp2 with Giardia exposure in Caco-2 and HT-29 cells. The protein expression of Skp2 with Giardia exposure in Caco-2 and HT-29 cells was detected by western blotting. (C) The qPCR and western blot analysis were used to measure mRNA and protein expression levels of Skp2, p21 and p27 in Caco-2 and HT-29 cells transfected with si-Skp2/si-NC for 48 h. All experiments were repeated at least three times. Data were presented as mean ± SD. *p < 0.05, **p < 0.01 versus relative control group. (B, C) The results of qPCR analyses (n = 3) were normalized against the level of GAPDH. (A–C) Representative pictures are shown.

Figure 6

Host cell cycle arrest induced by Giardia via ROS‐mediated ER stress. (A) The ROS assay kit was used to measure ROS generation in Caco-2 cells with Giardia exposure for 0, 3, 6, 12 h (n = 3 wells/group, scale bar = 100 μm). Under treatment of the combination of Giardia with ROS inhibitor NAC in Caco-2 cells, (B) cell cycle distribution was detected by flow cytometry, (C) the expressions of UPR-related proteins, p21, p27, cyclins, CDKs and E2Fs were detected by western blotting, and formation of E2F1-RB complex was detected by co-IP. The results of western blot analyses (n = 3) were normalized against the level of β-actin. All experiments were repeated at least three times. Data were presented as mean ± SD. *p < 0.05, **p < 0.01 versus relative control group. (A–C) Representative pictures are shown. G0/G1, S, and G2 indicate the different phases of the cell cycle. Gl, Giardia.

Figure 7

Host cell apoptosis was regulated by Giardia exposure via activating three UPR signaling pathways. (A) Western blot analysis was used to measure the protein levels of cleaved CASP3 and cleaved PARP in Caco-2 cells treated with or without inhibitors accompanied by Giardia exposure for 6 h; IRE1 inhibitor (MKC3946), PERK inhibitor (GSK2606414) and ATF6 inhibitor (AEBSF). (B) Apoptotic effects on Caco-2 cells during Giardia exposure under application of inhibitors (MKC3946, GSK2606414 or AEBSF) as assessed by AO/EB staining (n = 3 wells/group, scale bar = 1000 μm). The fluorescence intensity was quantified using Image J. Under treatment of the combination of IRE1 inhibitor MKC3946 with Giardia in Caco-2 cells, (C) western blot analysis was used to measure the phosphorylation levels of AKT and JNK. (D) The phosphorylation levels of AKT and JNK were detected by western blotting in Caco-2 and HT-29 cells with Giardia exposure for 0, 3, 6, 12 h. Under treatment of the combination of Giardia with AKT inhibitor MKC2206 2HCl or JNK inhibitor SP600125, (E) the expression levels of cleaved CASP3 and cleaved PARP in Caco-2 and HT-29 cells were detected by western blot analysis, (F) AO/EB staining was used to detect cell apoptosis. The fluorescence intensity was quantified using Image J. All experiments were repeated at least three times. (A, C–E) The results of western blot analyses were normalized against the level of β-actin. Data were presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01 versus relative control group. (A–F) Representative pictures are shown. Gl, Giardia.

Figure 8

Schematic diagram illustrating the molecular mechanism of cell cycle arrest and apoptosis regulated by ER stress during Giardia exposure (by Figdraw, https://www.figdraw.com).